Olympus IX81 Deconvolution System.

Volocity SOP

Fluorescence microscopy is extremely powerful tool due to its ability to show specifically labelled structures within a biological sample. However in wide field fluorescent microscopy the images collected  always contain some in focus and out of focus light. This means that an image of a certain structure is always blurred by the light that is present from structures above and below the focal plane. This results in  a loss of  image contrast and resolution.

This process is due to the optical properties of light and how that light behaves as it passes through the different optical components in the microscope system. When Light is  emitted from any point in the specimen a fraction of the light is collected by the objective and focused in the corresponding point in the microscope image plane. However the image of the point of light does not form a point in the image plane. Instead it produce a diffraction pattern of concentric rings of light surrounding a central disk (see image below). This disc is called an  an Airy disc. The radius of  this disk is determined by the NA of the objective lens. Higher NA objectives have a greater resolving power.

The  point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from a point source in the specimen and transmitted to the image plane. The point spread function is a well defined physical property and can be determined accurately for most imaging systems.   Deconvolution uses the knowledge we have on this point spread function to reverse the process and reassign the light to the correct image plane. Deconvolution microscopy uses computer-based  algorithms to remove the out of focus blur associated with wide field fluorescent microscopy and improve resolution.

Below: 3D image of Dicty microtubules GFP-Tubulin. Convolved and deconvolved. (Images captured on the Olympus BX81 Deconvolution Microscope using Volocity software.Images provided by Jason King R6)

    

Below Dicty mitochondria images using a mitochondrial targeted GFP.

                           


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Last modified: 02/25/16